The goal of the project is to understand the mechanism by which proteolytic enzymes, particularly thrombin, react with alpha-2-macroglobulin (alpha-2-M) and to understand the effect of the binding of other proteins. The biologic significance rests both with the importance of the clearance of proteases as a protective mechanism, and the need to define other functions of alpha-2-M which current work suggests may be extensive. The reaction with proteases will be studied by rapid-mixing stop-flow and conventional kinetic methods. Total enzyme binding, formation of covalent complexes, appearance of new thiol groups, and conformational changes, will be monitored in parallel in order to define the causal relation between these events and to develop a mechanistic model. For the slow reactions, the kinetics of complexes measured on gel electrophoresis will be studied. Of particular interest are several high molecular weight complexes which are believed to represent multivalent species in which several alpha-2-M chains are cross-linked via covalent bonds to a single enzyme molecule. The structure and function of this protein will also be studied by immunologic methods; the ability of members of a library of monoclonal antibodies to discriminate native and covalently modified alpha-2-M and its protease complexes will be investigated. Finally, a search for reactive groups, and structure-activity studies with amine donor molecules, will be undertaken to define the possible relation between this protein and the transglutaminases. The most obvious biological function of alpha-2-M is the clearance of proteolytic enzymes from the circulation, but there is suggestive evidence for its involvement in several other physiologic areas. Alpha-2-M is a substrate for Factor XIIIa and binds to platelet-derived growth factor. In addition, peptides derived from alpha-2-M have immunosuppressive and tumor-regulating activity. One of the reactive centers of alpha-2-M is a recently-discovered configuration of amino acids, an internal cys-glu thiolester, also found in proteins of the complement system. This may be important as a general mechanism by which proteins bind to other macromolecules.